Culture medium for detecting bacteria of listeria genus

ABSTRACT

The invention concerns a culture medium for isolating bacteria of Listeria genus, in particular  L. monocytogenes , characterised in that it comprises in a Listeria culture medium, at least a specific agent for identifying γ-mannosidase for use in solid medium.

[0001] The present invention relates to a chromogenic culture mediumintended to demonstrate bacteria of the Listeria genus, in particularListeria monocytogenes.

[0002] Both in the clinical field and in the agrofoods field,investigating Listeria, and in particular Listeria monocytogenes, isincreasingly important, since these bacteria are often disseminated inagrofood products and lead to serious infections in sensitive patients(pregnant women, elderly individuals, etc.).

[0003] In fact, for a few years, governments have been setting upincreasingly strict surveillance networks, in particular for products ofthe agrofoods industry. Thus, a generalization of the epidemiologicalsurveillance of Listeria monocytogenes is observed.

[0004] It is therefore important to have a reliable and rapid test fordetecting contaminations with these bacteria, which test must be bothsensitive and specific.

[0005] There exist today certain culture media for detecting Listeria,such as PALCAM or OXFORD medium which are more or less selective, whichmake it possible to detect the presence of Listeria in samples. Theresults obtained using these media can exhibit certain inaccuraciesimplicating the need to carry out other tests to confirm the presence ofL. monocytogenes.

[0006] These media are in fact based on a combination of antimicrobialagents providing selectivity and the detection of enzymes providingspecificity. However, both the selectivity and the specificity of thesemedia can be improved, since they bring about in particular the presenceof many false positives or false negatives. A disadvantage of thesemedia is that they do not make it possible to distinguish the species L.monocytogenes.

[0007] Media also exist which include a test based on the detection ofphospholipases in order to directly detect L. monocytogenes by specificcoloration of the colony or by a specific halo surrounding the colony.However, these tests are also positive for L. ivanovii, which is alsophospholipase+in nature.

[0008] It was also suggested to differentiate L. monocytogenes from L.ivanovii in an identification step following the isolation step, usingin particular a test based on a difference in an “aminopeptidase”phenotype.

[0009] For the purposes of differentiating L. monocytogenes from L.ivanovii, the present invention is based on the detection ofalpha-mannosidase, a characteristic which is positive for L.monocytogenes and negative for L. ivanovii. This characteristic can, forexample, be revealed on pure cultures using the enzyme substratenitrophenyl-α-mannoside, which is colorless and which releases ayellow-colored nitrophenyl when the test is positive.

[0010] The present invention demonstrates that it is possible to revealthe α-mannosidase in colonies on solid medium by detecting theα-mannosidase with a chromogenic or fluorogenic substrate in the solidmedium. The use of a substrate for α-mannosidase in a solid medium,derived for the first time in the present application, in particular hasthe advantage of preparing solid media in which it is possible todifferentiate L. monocytogenes from L. ivanovii as soon as isolation hasbeen carried out, without having to carry out further tests.

[0011] Thus, the present invention relates to a novel culture medium fordetecting and/or discriminating bacteria of the Listeria genus,characterized in that it comprises, in a Listeria culture medium, atleast one specific agent for identifying α-mannosidase which can be usedin solid medium. Said specific agent for identifying α-mannosidase whichcan be used in solid medium is preferably a chromogenic or fluorogenicagent chosen from α-mannosidase substrates.

[0012] The chromogenic agent which is an α-mannosidase substratepreferably comprises a precipitatable chromophore which is released byhydrolysis of the substrate by its enzyme. Thus, the bacterial colonybecomes colored as a function of the chromophore released, thechromophore released being solid in the culture medium, and thereforeremaining localized at the colony where it was released.

[0013] Preferably, said chromophore is chosen from the indoxyl,haloindoxyl (bromoindoxyl, chloroindoxyl, fluoroindoxyl, iodoindoxyl,dichloroindoxyl, chlorobromoindoxyl, trichloroindoxyl), methylindoxyl orhydroxyquinoline derivatives. Preferred derivatives are in particularchosen from the following derivatives: 6-chloroindoxyl, 5-bromoindoxyl,3-bromoindoxyl, 6-fluoroindoxyl, 5-iodoindoxyl, 4,6-dichloroindoxyl,6,7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl,4,6,7-trichloroindoxyl, N-methylindoxyl and 8-hydroxyquinoline.

[0014] When said α-mannosidase substrate is coupled to a fluorophore,4-methylumbelliferyl is preferably used.

[0015] Preferably, the α-mannosidase substrate is indoxyl-α-mannoside.The concentration of the chromogenic agent in the medium is betweenapproximately 0.01 and 0.5 g/l. A preferred concentration is 0.05 g/l.

[0016] It may also be advantageous to add a colored-reaction activator,to the medium, in order to improve the quality of the reaction observed.A suitable activator for such a use is methyl-α-mannoside, which isintegrated into the medium at a concentration of between approximately0.01 and 0.5 g/l, preferably 0.25 g/l.

[0017] The medium according to the present invention thus makes itpossible to detect L. monocytogenes, and to discriminate this bacteriumwith respect to L. ivanovii, without carrying out any additional test.In order to optimize the results given by the medium according to theinvention, it may be advantageous to add factors selective for Listeria,as used in the media of the prior art. A medium specific for L.monocytogenes is therefore obtained.

[0018] A subject of the invention is also the use of a culture mediumaccording to the invention, for detecting and/or discriminating bacteriaof the Listeria genus, in particular L. monocytogenes.

[0019] A subject of the present invention is also a method for detectingand/or discriminating bacteria of the Listeria genus, in a sample,characterized in that it comprises the following steps:

[0020] a. inoculating a culture medium according to the invention withsaid sample or an inoculum derived from the sample,

[0021] b. detecting the presence of bacteria of the Listeria genus onsaid culture medium,

[0022] c. optionally, differentiating L. monocytogenes from L. ivanoviiand from the other Listeria present on said culture medium.

EXAMPLES Example 1

[0023] A preferred medium for implementing the invention comprises (perliter): agar 10 g calf brain infusion 12.5 g proteose peptone 10 g beefheart infusion 5 g sodium chloride 5 g Na₂HPO₄ 4 g KH₂PO₄ 2 g glucose 2g lithium chloride 7.5 g lecithin 3 g ofloxacin 0.0004 g colistin 0.015g ceftazidime 0.0134 g methyl-α-mannoside 0.25 g indoxyl-α-mannoside0.05 g

Example 2

[0024] The plating out of bacteria on the medium according to theinvention gave the following results (incubation for 24 hours at 37°C.). Halo Colony center L. monocytogenes white blue L. ivanovii whitecolorless other Listeria no halo —

[0025] The use of the medium according to the invention therefore makesit possible to detect and discriminate L. monocytogenes, L. ivanovii andthe other species of Listeria.

1. A culture medium for detecting and/or discriminating bacteria of theListeria genus, characterized in that it comprises, in a Listeriaculture medium, at least one specific agent for identifyingα-mannosidase which can be used in solid medium.
 2. The culture mediumas claimed in claim 1, characterized in that said agent specific foridentifying α-mannosidase which can be used in solid medium is achromogenic or fluorogenic agent chosen from α-mannosidase substrates.3. The culture medium as claimed in claim 2, characterized in that saidchromogenic agent releases, by hydrolysis, a precipitatable chromophorechosen from the indoxyl, haloindoxyl (bromoindoxyl, chloroindoxyl,fluoroindoxyl, iodoindoxyl, dichloroindoxyl, chlorobromoindoxyl,trichloroindoxyl), methylindoxyl or hydroxyquinoline derivatives, inparticular the following derivatives: 6-chloroindoxyl, 5-bromoindoxyl,3-bromoindoxyl, 6-fluoroindoxyl, 5-iodoindoxyl, 4,6-dichloroindoxyl,6,7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl,4,6,7-trichloroindoxyl, N-methylindoxyl or 8-hydroxyquinoline.
 4. Theculture medium as claimed in claim 2, characterized in that thefluorophore agent linked to the α-mannosidase substrate is4-methylumbelliferyl.
 5. The culture medium as claimed in one of claims1 to 4, characterized in that said medium also contains acolored-reaction activator.
 6. The culture medium as claimed in claim 5,characterized in that said activator is methyl-α-mannoside.
 7. Theculture medium as claimed in one of claims 1 to 6, characterized in thatit also comprises factors selected for Listeria.
 8. The culture mediumas claimed in one of claims 1 to 7, characterized in that theα-mannosidase substrate is indoxyl-α-mannoside.
 9. The culture medium asclaimed in one of claims 1 to 8, characterized in that it comprises (perliter): agar 10 g calf brain infusion 12.5 g proteose peptone 10 g beefheart infusion 5 g sodium chloride 5 g Na₂HPO₄ 4 g KH₂PO₄ 2 g glucose 2g lithium chloride 7.5 g lecithin 3 g ofloxacin 0.0004 g colistin 0.015g ceftazidime 0.0134 g methyl-α-mannoside 0.25 g indoxyl-α-mannoside0.05 g


10. The culture medium as claimed in one of claims 1 to 9, characterizedin that it is specific for L. monocytogenes.
 11. The use of a culturemedium as defined in one of claims 1 to 10, for detecting and/ordiscriminating bacteria of the Listeria genus.
 12. A method fordetecting and/or discriminating bacteria of the Listeria genus in asample, characterized in that it comprises the steps consisting in: a.inoculating a culture medium as defined in one of claims 1 to 10 withsaid sample or an inoculum derived from the sample, b. detecting thepresence of bacteria of the Listeria genus on said culture medium, c.optionally, differentiating L. monocytogenes from L. ivanovii and fromthe other Listeria present on said culture medium.